This project is directed at developing and using physical and mathematical methodologies to understand integrative aspects of complex cellular processes. Emphasis has been on studying the functioning of multicomponent, supramolecular entities such as composite membranes and elements of cytoskeletal networks, particularly to understand their involvement in cellular organization and cell shape. Related in vitro reconstitution studies of purified cell components also have been undertaken. These investigations oftentimes employ unusual physical instrumentation, in part to examine the general utility of such devices for biological research. For example, a fluorescence correlation microscope (FCM) recently has been constructed which now is being used to investigate the sizes and stability of aberrant tubulin ring polymers induced by dolastatin, cryptophycin, and similar antimitotic agents. This study is of intrinsic interest, as these ring-forming compounds currently are being evaluated elsewhere as possible anti-tumor drugs, and it also serves to calibrate the instrument and drive development of new theory for interpreting measurements. We recently began studies of the structure and stability of protein-polysaccharide conjugates that are being developed as vaccines against enteric pathogens, the objective being to determine the sizes of vaccine conjugates when diluted to low physiological concentrations in a range not easily accessible to study by other physical techniques. Similar studies of other multi-component supramolecular assemblies are planned, among which are investigations of the size distributions of reconstituted clathrin baskets. In the latter case, measurements will be related to molecular parameters for the interaction energies of basket constituents, using physical analyses which we recently published. To infer possible triggering mechanisms for endocytosis and related intracellular membrane vesicle-forming processes, we have carried out mathematical and computational analysis of complex pathways of phosphatidylinositide biosynthesis and metabolism, showing that coupling of enzymatic activity to vesicle curvature can introduce nonlinearities and feedback loops leading to pseudo-bistability and triggering. In yet other studies we have used small angle neutron scattering (SANS) and ultra small angle x-ray scattering (USAXS) to study the supramolecular structures of tubulin polymers in solution, including isolated rings and higher order ring aggregates that appear when tubulin is acted upon by dolastatin. Preliminary evidence indicates that the aggregates are composed of columnar stacks of elemental, individual rings.